ABSTRACT Eastern (EEEV), Venezuelan (VEEV), and Western (WEEV) equine encephalitis viruses are mosquito-transmitted alphaviruses with the potential to cause fatal neuroinvasive disease in humans and domesticated animals. These viruses also can be spread by the aerosol route and thus, have significant potential as biowarfare/bioterrorism agents. In 2019, EEEV human infections increased to levels not seen since the 1950s. Currently, there are no antiviral therapies or vaccines licensed for these alphaviruses. The existing investigational live-attenuated VEEV vaccine (LAV; TC-83) was generated >40 years ago, is highly reactogenic, poorly immunogenic, and causes disease in up to 20% of recipients. For EEEV and WEEV, investigational formalin-inactivated vaccines have been given to at-risk workers. While both elicit antibody responses, they provide uncertain protection and require multiple booster injections. A trivalent veterinary vaccine against VEEV, EEEV and WEEV is available but is so poorly immunogenic that domestic animals must be boosted annually and disease is seen in vaccinated animals. Here, we propose to develop safe and effective LAVs vaccine against these alphaviruses using recent advances in understanding of the virulence mechanisms of each. These advances will allow mutagenesis of pathogenicity domains that specifically reduce virulence, increase cytokine induction and antigen presentation and provide long-lasting protection. Furthermore, the mechanism of action of each mutation will be known and each will be specifically designed to resist reversion. We will combine mutations to create an optimal vaccine strain for each virus and then investigate their use in multivalent formulations, critical to veterinary vaccine development and highly desired for human formulations. We will identify correlates of protection for each vaccine and address the extent of cross-protection versus encephalitic viruses. For the first time with alphaviruses, the individual and combined effects of mutations with known mechanisms of action will be tested for effects on virus replication, disease and immunogenicity in animals.